Fig. 1 CpG induces the expression of OX40 on CD4 T cells.

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Fig. 1 CpG induces the expression of OX40 on CD4 T cells. CpG induces the expression of OX40 on CD4 T cells. (A) A20 tumor–bearing mice were treated either with vehicle (top) or CpG (middle). Forty-eight hours later, tumors were excised and a single-cell suspension was stained and analyzed by flow cytometry. (B) OX40 expression within the CD3+CD4+ subset was separately analyzed for FoxP3-negative [effector T cell (Teff)] and FoxP3-positive [regulatory T cell (Treg)] subsets. Fold changes of OX40+ cells were calculated according to their frequencies in the vehicle versus CpG treatment (n = 2). (C) Fine needle aspirates from CpG-injected and noninjected tumors of a follicular lymphoma patient were obtained 22 hours after treatment. Fluorescence-activated cell sorting (FACS) plots of OX40 expression within the CD4+ subset after a 24-hour rest in media. Top: Nontreated lesion. Bottom: CpG-treated site (n = 2). (D) Single-cell suspensions from biopsy specimens of human lymphoma (five mantle cell lymphomas and five follicular lymphomas) were exposed in vitro to CpG for 48 hours and analyzed for OX40 expression as in (B). (E) CpG-stimulated human lymphoma–infiltrating CD4+ T cells, CD8+ T cells, and CD19+ B cells were gated and visualized in tSNE (t-Distributed Stochastic Neighbor Embedding) space using Cytobank software. The viSNE map shows the location of each CD4+, CD19+, and CD8+ cell population (green, blue, and orange, respectively; bottom). Cells in the viSNE maps were colored according to the intensity of OX40 expression. CpG up-regulation of OX40 expression on a subset of CD4+ T cells is highlighted by a red box. (F) BALB/c mice were implanted subcutaneously with A20 lymphoma cells (5 × 106) on both the right and left shoulders. When tumors reached between 0.7 and 1 cm in the largest diameter (typically on days 8 to 9 after inoculation), phosphate-buffered saline and CpG (50 μg) were injected into one tumor site (left tumor). Sixteen hours later, 64Cu-DOTA-OX40 was administered intravenously via the tail vein. Positron emission tomography imaging of mice was performed 40 hours after in situ treatment. Left: Vehicle-treated. Right: CpG-treated. These images are representative of six mice per group. (G) Fresh A20 tumors were excised from animals (typically 5 to 6 days after inoculation), and either whole tumors (left), T cells purified from the tumor (middle), or whole tumor depleted of CD11b- and CD11c-expressing cells (right) were treated for 48 hours with media (top) or CpG (bottom) and were analyzed for their expression of OX40 by flow cytometry. (H) Left: A20 tumors were excised as in (F). Right: Single-cell suspensions from biopsy specimens of human follicular lymphoma. Tumors were treated for 48 hours with media and CpG with or without antibodies (1 μg/ml) to interleukin-2 (IL-2), IL-4, IL-10, granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-12, interferon-γ (IFN-γ), or tumor necrosis factor–α (TNF-α) and were analyzed for their expression of OX40 by flow cytometry. α–IL-12, *P = 0.0144; α–IFN-γ, **P = 0.0032; α–TNF-α, **P = 0.008, unpaired t test, either depleting antibody versus CpG alone. Idit Sagiv-Barfi et al., Sci Transl Med 2018;10:eaan4488 Published by AAAS